Zhang et al BMC Genomics (2021) 22:221 https://doi.org/10.1186/s12864-021-07479-4 RESEARCH ARTICLE Open Access Genome-wide identification and expression analysis of the B-box transcription factor gene family in grapevine (Vitis vinifera L.) Xiuming Zhang1,2†, Li Zhang1,2†, Miaomiao Ji1,2, Yifei Wu1,2, Songlin Zhang1,2, Yanxun Zhu1,2, Jin Yao1,2, Zhi Li1,2, Hua Gao1,2* and Xiping Wang1,2* Abstract Background: B-box (BBX) zinc-finger transcription factors play important roles in plant growth, development, and stress response Although these proteins have been studied in model plants such as Arabidopsis thaliana or Oryza sativa, little is known about the evolutionary history or expression patterns of BBX proteins in grapevine (Vitis vinifera L.) Results: We identified a total of 25 VviBBX genes in the grapevine genome and named them according to the homology with Arabidopsis These proteins were classified into five groups on the basis of their phylogenetic relationships, number of B-box domains, and presence or absence of a CCT domain or VP motif BBX proteins within the same group showed similar exon-intron structures and were unevenly distributed in grapevine chromosomes Synteny analyses suggested that only segmental duplication events contributed to the expansion of the VviBBX gene family in grapevine The observed syntenic relationships between some BBX genes from grapevine and Arabidopsis suggest that they evolved from a common ancestor Transcriptional analyses showed that the grapevine BBX genes were regulated distinctly in response to powdery mildew infection and various phytohormones Moreover, the expression levels of a subset of BBX genes in ovules were much higher in seedless grapevine cultivars compared with seeded cultivars during ovule development, implying a potential role in seed abortion Additionally, VviBBX8, VquBBX15a and VquBBX29b were all located in the nucleus and had transcriptional activity except for VquBBX29b Conclusions: The results of this study establish the genome-wide analysis of the grapevine BBX family and provide a framework for understanding the biological roles of BBX genes in grapevine Keywords: Grapevine, BBX family, Transcription factors, Expression profile Background Transcription factors (TFs) play varied and important roles in plant growth, development and biological responses [1] Zinc-finger TFs are one of the most populous classes of TFs in plants, and can be classified into several families based on the number and location of characteristic amino acid sequence motifs [2] The * Correspondence: gaohua2378@163.com; wangxiping@nwsuaf.edu.cn † Xiuming Zhang and Li Zhang contributed equally to this work State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China Full list of author information is available at the end of the article B-box (BBX) zinc-finger TFs contain one or two conserved domains of approximately 40 amino acids near the amino terminus, and may also contain a CCT (CONSTANS, CO-like and TOC1) domain and/or a valine-proline (VP) motif at the carboxyl terminus [3] In Arabidopsis thaliana (Arabidopsis), BBX genes have been identified and classified into five subfamilies based on presence of these domains [4] Additionally, it has been reported that the conserved B-box domain mediates protein-protein interactions, while the CCT domain functions in transcriptional regulation [5, 6] © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Zhang et al BMC Genomics (2021) 22:221 BBX proteins participate in myriad biological processes in plants In Arabidopsis, AtBBX1, AtBBX4, AtBBX7 and AtBBX32 regulate photoperiodic flowering [7–10] Meanwhile, similar flowering roles have been reported in rice [11], barley [12], sorghum [13] and Chinese Cabbage [14] Other studies have found that several AtBBX proteins participate in seedling photomorphogenesis through the HY5-COP1 regulatory module [15–17] For instance, the HY5 transcription factor directly binds to a G-box cis-element present in the promoters of AtBBX30 and AtBBX31 and represses their expression, thus negatively regulating photomorphogenesis [18] In rice, OsBBX14 promotes photomorphogenesis by directly binding the T/G-box cis-element of the OsHY5L1 promoter under blue light conditions [19] In pear, PpBBX16 (the homolog of AtBBX22) and PpHY5 jointly activate the expression of PpMYB10 and other structural genes to positively regulate light-induced anthocyanin accumulation [20] In apple, MdBBX20 integrates the influence of ultraviolet radiation and low temperature to promote the accumulation of anthocyanin [21] In addition, BBX proteins have also been found to participate in response to environmental stress For example, AtBBX31 promotes tolerance to UV-B radiation in Arabidopsis [22], and CmBBX22 regulates leaf senescence in chrysanthemum [23] Heterologous expression of apple MdBBX10 in Arabidopsis enhances tolerance to salt [24] In grapevine, VvCOL and VvCOL1 (VviBBX2 and VviBBX5, respectively) participate in flowering and bud dormancy [25] Grapevine (Vitis vinifera L.) is one of the most economically important perennial fruit crops throughout the world Grapes can be consumed fresh or dried, or can be processed into juice, wine, and jam Seedless grapevine cultivars are particularly important, especially for fresh and dried fruit However, worldwide production of both seeded and non-seeded grapes is increasingly limited by biotic and abiotic stresses Cultivar improvement through traditional breeding and biotechnology is an exciting prospect, but options have been limited by the general lack of knowledge about key genes that mediate stress responses Fortunately, the publication of a draft grapevine genome [26] has facilitated the identification of transcription factors In this study, we identified members of the BBX family in grapevine from the draft genome sequence and gained insight into their potential function based on gene and protein structure, phylogeny, synteny, subcellular localization and transcriptional activity, as well as expression during ovule development, in response to pathogen challenge and various phytohormones Taken together, this work will be helpful for future studies of BBX gene functions in grapevine Page of 16 Results Identification and characterization of grapevine BBX genes To identify BBX genes in the grapevine genome, we employed a Hidden Markov Model (HMM)-based approach and the amino acid sequence profile of the Bbox-type zinc-finger domain (Pfam; PF00643) The resulting protein sequences were assessed for the presence of a B-box domain as defined by the Simple Modular Architecture Research Tool (SMART; http:// smart.embl-heidelberg.de/) and the Conserved Domain Database (CDD; https://www.ncbi.nlm.nih gov/ Structure/cdd/cdd.shtml) This resulted in the identification of 25 putative BBX genes For the sake of nomenclature and consistency, these were designated as VviBBXs (Table 1, Additional file 1: Text S1), based on the recently proposed grapevine nomenclature system [27] The length of the encoded proteins ranged from 127 to 469 amino acids, and their predicted molecular mass ranged from ~ 14.3 to 50.9 kDa The isoelectric points of the predicted proteins ranged from ~ 4.1 to 8.7 (Table 1) Phylogeny and conserved domains of the grapevine BBX proteins To analyze the evolutionary relationship and potential functional divergence of the VviBBX gene family, a total of 205 BBX proteins, including 32 from Arabidopsis, 29 from tomato, 30 from rice, 64 from apple and 25 from pear, were used to construct a phylogenetic tree (Fig 1, Additional file 2: Text S2) This resolved the grapevine BBX proteins into five clades which mostly corresponded to their assigned structural groups (Table 1, Fig 3a) However, an exception was found in the above clades The VviBBX27 protein was presumptively phylogenetically in clade IV based on the structure group, but it was located in phylogenetic clade V (Fig 1) As shown in the phylogeny tree, it is evident that BBX genes of the woody plants (grapevine, apple and pear) clustered together And most of the grapevine BBXs also clustered together with proteins from Arabidopsis and tomato, instead of rice, consistent with the closer relationship of grapevine to the two eudicots The conserved sequences of the B-box1 and B-box2 zinc finger domains were C-X2-C-X7–8-C-X2-D-X-A-XL-C-X2-C-D-X3-H-X2-N-X4-H and C-X2-C-X8-C-X7-CX2-C-X4-H(N)-X6–8-H, respectively In addition, the CCT domain of twelve of the grapevine proteins with the form of R-X5-R-Y-X2-K-X3-R-X3-K-X2-R-Y-X2-R-KX2-A-X2-R-X-R-X2-G-R-F-X-K was highly conserved A graphical representation of amino acid conservation with these motifs is shown in Fig Alignment of the protein sequences revealed that the B-box1 domain was more conserved than the B-box2 as a result of five absolutely 394 469 449 432 Vitvi10g00219.t01 VIT_200s0194g00070.1 GSVIVT01003473001 XP_002265377.2 1245 Vitvi19g00408.t01 VIT_219s0014g05120.1 GSVIVT01014591001 XP_010644324.1 1113 Vitvi12g00542.t01 VIT_212s0059g02500.1 GSVIVT01030628001 XP_019078853.1 1305 Vitvi07g00252.t01 VIT_207s0104g01360.1 GSVIVT01010991001 XP_002268490.1 1185 Vitvi01g01729.t01 VIT_201s0146g00360.1 GSVIVT01000951001 XP_002263613.1 1410 VviBBX8 VviBBX9 VviBBX10 VviBBX11 VviBBX12a VviBBX12b Vitvi14g01487.t01 VIT_214s0068g01380.1 GSVIVT01033017001 XP_010660698.1 1350 VviBBX15a XP_002276181.1 1233 299 298 Vitvi12g02441.t01 VIT_212s0134g00400.1 GSVIVT01000440001 XP_002272924.1 900 VviBBX29a VviBBX29b Vitvi19g00322.t01 VIT_219s0014g03960.1 GSVIVT01014471001 XP_002284274.1 897 RVX03703.1 Vitvi09g01361.t01 VIT_209s0054g00530.1 – VviBBX32 260 39946.58 26443.94 31682.56 22812.56 33157.15 33371.52 20366.2 23371.44 45929.41 47784.66 49441.06 50856.79 43918.32 47040.03 41137.3 45041.34 44982.85 38993.62 38001.35 42699.57 chr9: 21166392–21167745 (+) chr12: 7296673–7297056 (−) chr19: 4194453–4195989 (−) chr12: 8048322–8050082 (+) Domains 28155.08 8.67 1BBOX 7.71 1BBOX 4.22 1BBOX 32739.41 14258.41 4.06 1BBOX 32306.61 4.43 1BBOX 4.76 2BBOX 4.61 2BBOX 4.87 2BBOX 5.05 2BBOX 6.65 2BBOX 7.79 2BBOX 6.87 2BBOX 6.40 2BBOX 5.29 1BBOX + CCT 5.40 1BBOX + CCT 6.09 2BBOX + CCT 5.89 2BBOX + CCT 6.22 2BBOX + CCT 7.68 2BBOX + CCT 6.33 2BBOX + CCT 5.14 2BBOX + CCT 4.76 2BBOX + CCT V V V V V IV IV IV IV IV IV IV IV III III II II II II II II II 6.75 2BBOX + CCT + VP motif I 6.24 2BBOX + CCT + VP motif I Nuclear Extracellular Nuclear Nuclear Nuclear Nuclear Extracellular Extracellular Nuclear Nuclear Nuclear Extracellular Extracellular Nuclear/ Cytoplasmic Nuclear Nuclear Nuclear Nuclear Nuclear/ Extracellular Nuclear Nuclear Nuclear Nuclear Nuclear Nuclear Structural Subcellular group localization 5.77 2BBOX + CCT + VP motif I MW (Da) pI chrUn: 11688677–11689572 (−) 23153.46 chr1: 3849170–3854088 (+) chr5: 23065597–23068639 (−) chr19: 356889–371959 (+) chr18: 34402834–34404843 (+) chr18: 11546101–11548030 (+) chr3: 310821–312269 (+) chr4: 19619474–19622085 (−) chr3: 594823–598603 (−) chr17: 3813174–3815238 (−) chr1: 3190849–3193178 (−) chr14: 25084927–25088384 (+) chr1: 23236072–23239673 (−) chr7: 2762240–2765564 (−) chr12: 7291808-7293953 (+) chr19: 5406770–5409091 (−) chr10: 2260168–2272241 (+) chr12: 9692684–9699362 (−) chr11: 19920787–19922322 (+) chr4: 7669506–7671340 (−) chr14: 22695952–22698379 (+) Abbreviations: CDS, coding sequence; aa, amino acid; chr, chromosome; Un, unknown chromosome; MW, molecular weight; pI, isoelectric point 783 XP_010657357.1 384 Vitvi12g00543.t01 VIT_212s0059g02510.1 – VviBBX30 127 357 639 212 RVW13823.1 Vitvi01g00346.t01 VIT_201s0011g04240.1 GSVIVT01011821001 XP_002279997.2 1074 Vitvi10g02328.t01 VIT_200s0203g00210.1 – VviBBX27 VviBBX28 293 239 Vitvi05g01519.t01 VIT_205s0102g00750.1 GSVIVT01010794001 XP_002268700.1 720 205 Vitvi18g02424.t01 VIT_218s0089g01280.1 GSVIVT01037095001 XP_019071822.1 618 VviBBX22a VviBBX22b Vitvi19g00031.t01 VIT_219s0014g00350.1 GSVIVT01014097001 XP_002283666.1 882 303 VviBBX21b Vitvi18g01048.t01 VIT_218s0001g13520.1 GSVIVT01009821001 XP_002280716.1 912 VviBBX25 302 184 210 410 370 414 415 Vitvi03g00026.t01 VIT_203s0038g00340.1 GSVIVT01024204001 XP_002274649.1 909 VviBBX21a 555 Vitvi03g00049.t01 VIT_203s0038g00690.1 GSVIVT01024173001 XP_002267957.1 633 – VviBBX19b Vitvi04g01423.t01 VIT_204s0023g03030.1 GSVIVT01018818001 RVW36633.1 VviBBX19a VviBBX15b Vitvi17g00328.t01 – Vitvi01g00288.t01 VIT_201s0011g03520.1 GSVIVT01011897001 XP_002282578.1 1299 434 Vitvi12g00757.t01 VIT_212s0057g01350.1 GSVIVT01030127001 XP_002264506.2 1248 VviBBX7 347 361 Vitvi04g00665.t01 VIT_204s0008g07340.1 GSVIVT01036037001 XP_002263458.1 1044 Vitvi11g01309.t01 VIT_211s0052g01800.1 GSVIVT01029107001 XP_002277953.1 1086 VviBBX5 391 CDS (bp) Protein (aa) Position VviBBX6 Accession no Vitvi14g01296.t01 VIT_214s0083g00640.1 GSVIVT01036499001 XP_002282509.1 1176 Locus ID VviBBX2 CRIBI v2.1 ID VCost.v3 ID Gene ID Table Detailed information of VviBBX gene family members in grapevine Zhang et al BMC Genomics (2021) 22:221 Page of 16 Zhang et al BMC Genomics (2021) 22:221 Page of 16 Fig Phylogenetic analysis of BBX proteins from grapevine, Arabidopsis, tomato, apple, pear and rice The tree was divided into five clades, which are marked by different colors and named as Clade I, II, III, IV and V The bootstrap values are indicated at each node conserved amino acid residues (two Asps, Ala, Leu and Asn) in all B-box1 domain (Additional file 3: Fig S1) Based on amino acid sequence conservation, number of B-box domains, and the presence or absence of the CCT domain, the 25 grapevine BBX proteins fell into five distinct structural classes (Table 1), which is consistent with previous results in Arabidopsis [4] Group I, comprising three of the proteins, contained two B-box domains and one CCT domain The seven representatives of Group II also contained two B-box domains and a CCT domain, but were distinguished from Group I based on the absence of the highly conserved amino acid sequence (SANPLARR) in the B-box2 domain and VP motif amino-terminal to the CCT domain seen in Group I proteins (Additional file 4: Fig S2) Group III, comprising two proteins, contained one B-box domain and one CCT domain The eight members of Group IV contained two B-box domains, while Group V proteins (five members) had only one B-box domain Analysis of conserved protein motifs and exon-intron structure of VviBBX genes To gain additional insight into the conservation and diversification of the grapevine BBX gene family, we analyzed the conserved protein motifs encoded by the genes, as well as exon-intron structures (Fig 3) Sixteen conserved motifs were identified (Fig 3b), with four corresponding to B-box1 (Motifs 1/5), B-box2 (Motif 3), and CCT (Motif 2) Interestingly, we observed that Motifs 8, 13, and 14 were present only in Group III, which might contribute to the functional divergence of BBX genes Motifs and were seen in all members of Group II, but also in VviBBX27 in Group IV, suggesting that VviBBX27 may have evolved from a Group II gene The motif sequences and logos are listed in Additional file 5: Table S1 Additionally, VviBBX22b was the longest BBX gene (14.3 Kb) We also found that three genes in Group V (VviBBX28, VviBBX30 and VviBBX32) had a single exon, while all others carried between two Zhang et al BMC Genomics (2021) 22:221 Page of 16 Fig Amino acid sequence conservation within the B-box and CCT domains of grapevine BBX proteins (a), (b) and (c) represent the amino acid sequence alignment of the B-box1, B-box2 and CCT domain, respectively The x axis indicates the amino acids present at each position, and the y-axis and height of each letter indicate the degree of conservation of each residue across all proteins and five exons Moreover, all the genes in Groups I, II and III contained three, four and two exons, respectively (Fig 3c) Chromosomal distribution and synteny analysis among VviBBX genes Based on their annotated genomic locations, the 25 VviBBX genes were found to be widely distributed among the grapevine chromosomes (Fig 4) Chromosome 12 contained the most VviBBX genes (four), whereas Chromosomes and 19 both possessed three genes, Chromosomes 3, 4, 14, and 18 had two VviBBX genes, and Chromosomes 5, 7, 9, 10, 11, and 17 had only one gene The chromosomal location of VviBBX28 was on the chromosome Unknown Segmental duplications and tandem duplications contribute to the evolution of gene families [28] According to Fig and Additional file 6: Table S2, four segmental duplication events have occurred: VviBBX9 to VviBBX10, VviBBX12a to VviBBX12b, VviBBX19a to VviBBX19b, and VviBBX21a to VviBBX21b However, no tandem duplication was observed according to the Fig Characterization of grapevine BBX genes a Phylogenetic analysis of BBX proteins in grapevine b Distribution of conserved motifs identified in the 25 VviBBX proteins Each motif is represented by a number in a colored box Detailed sequence information for each motif is shown in Additional file 5: Table S1 c Exon-intron structure of grapevine BBX genes Exons are represented by pink boxes and black lines connecting two exons represent an intron The Roman numerals (I-V) indicate the five structural groups Zhang et al BMC Genomics (2021) 22:221 Page of 16 Fig Distribution and synteny analysis of VviBBX genes on grapevine chromosomes The approximate chromosomal locations of the BBX genes are indicated on the periphery The colored lines linking genes from different chromosomes denote segmental duplication events foregoing descriptions of Holub [29], and thus only segmental duplication seems to have taken part in the evolution of the grapevine BBX gene family To gain insight into the evolutionary relationship between VviBBX and AtBBX genes, we analyzed genomic synteny A total of 26 gene pairs, comprising 17 VviBBXs and 23 AtBBXs, were identified (Fig 5, Additional file 7: Table S3) Among those, we found nine orthologous pairs, and also identified eight orthologous gene pairs with one grapevine gene corresponding to multiple Arabidopsis genes We noted that AT2G32310 was not included in the Arabidopsis BBX family, but contained a CCT domain which was also found in VviBBX10 Finally, three orthologous gene pairs where multiple grapevine genes corresponded to a single Arabidopsis gene were found (Additional file 7: Table S3) In brief, these syntenic relationships suggest that about two-thirds of the BBX genes appeared before the divergence of grapevine and Arabidopsis Zhang et al BMC Genomics (2021) 22:221 Page of 16 Fig Synteny analysis of BBX genes between grapevine and Arabidopsis The chromosomes of grapevine and Arabidopsis are arranged as a circle Syntenic occurrences of BBX genes are represented by colored lines To investigate potential selective pressure for VviBBX gene duplication events, we calculated the nonsynonymous (Ka) and synonymous (Ks) substitution rates Between grapevine and Arabidopsis, or grapevine alone, all segmentally duplicated gene pairs showed Ka/Ks ratios of < 1, suggesting that they had evolved primarily under purifying selection The divergence time of the segmental duplication event was calculated as between ~ 77 and 110 million years ago (Mya) in grapevine alone (Additional file 6: Table S2), and between ~ 102 and 349 Mya, with an average of 178.8 Mya, in grapevine and Arabidopsis (Additional file 7: Table S3) VviBBXs gene expression profiles in response to E necator inoculation and hormone treatments To help identify a possible function of the VviBBX genes in response to powdery mildew, we inoculated healthy plants of the powdery-mildew resistant genotype ‘Shang24’ with Erysiphe necator, the causative agent of grapevine powdery mildew, and monitored the expression of the 25 VviBBX genes by semi-quantitative RTPCR (Real-time polymerase chain reaction) Within 12 h after inoculation, the expression levels of ten genes (VviBBX2, VviBBX8, VviBBX11, VviBBX12b, VviBBX21a, VviBBX22a, VviBBX22b, VviBBX28, VviBBX29a and VviBBX29b) were up-regulated, while those of seven ... 4.61 2BBOX 4.87 2BBOX 5.05 2BBOX 6.65 2BBOX 7.79 2BBOX 6.87 2BBOX 6.40 2BBOX 5.29 1BBOX + CCT 5.40 1BBOX + CCT 6.09 2BBOX + CCT 5.89 2BBOX + CCT 6.22 2BBOX + CCT 7.68 2BBOX + CCT 6.33 2BBOX +... inoculation, the expression levels of ten genes (VviBBX2, VviBBX8, VviBBX11, VviBBX1 2b, VviBBX21a, VviBBX22a, VviBBX2 2b, VviBBX28, VviBBX29a and VviBBX2 9b) were up-regulated, while those of seven ... (Table 1) Phylogeny and conserved domains of the grapevine BBX proteins To analyze the evolutionary relationship and potential functional divergence of the VviBBX gene family, a total of 205 BBX